KR100573424B1 - Immune milk containing Helicobacter pylori antibody ? its manufacturing method - Google Patents

Abstract

The present invention relates to immune milk containing an anti- Helicobacter pylori antibody and a method for producing the same, and more specifically, to Helicobacter pylori, which is a causative agent of gastritis and gastric ulcer, as an antigen. The present invention relates to an immunomilk containing an anti- Helicobacter pylori antibody and a method of producing the same, to produce an immunomilk containing specific antibodies to be used as a dietary supplement without side effects.

The present invention as described above,

Passage the Helicobacter pylori strains at 37 ° C. in a 10% CO ₂ incubator at 2-3 days with a sheep blood agar (SBA) plate;

Culturing the above strain in liquid at 37 ° C. for 48 hours in a Mueller Hinton Broth (MHB) medium containing 5% FBS (fetal bovine serum) inactivated at 56 ° C. for 30 minutes;

Sterilization with 0.5% formalin solution after the culture;

Recovering the bacteria by centrifugation at 4,000 rpm for 30 minutes at 4 ° C;

Centrifuging the recovered bacteria with sterile PBS (phosphate buffered saline, pH 7.2) at 4,000 rpm for 30 minutes at 4 ° C., followed by washing and sonicating;

Preparing a vaccine by emulsifying the antigen in 1: 1 ratio with an antigen solution adjusted to O.D. = 1.0 at 667 nm with a spectrophotometer;

Intramuscularly injecting the prepared antigens two or more times eight times at two week intervals to the milking cow;

Milking from the milking cow; characterized in that it comprises a method for producing an immune milk, including an anti- Heliobacter pylori antibody, and the immune milk obtained thereby.

Helicobacter Pylori, Immune Milk

Description

Immune milk containing Helicobacter pylori antibody and its production method {Immune milk containing Helicobacter pylori antibody & its manufacturing method}

The present invention relates to immune milk containing an anti- Helicobacter pylori antibody and a method for producing the same, and more specifically, to Helicobacter pylori, which is a causative agent of gastritis and gastric ulcer, as an antigen. The present invention relates to an immunomilk containing an anti- Helicobacter pylori antibody and a method of producing the same, to produce an immunomilk containing specific antibodies to be used as a dietary supplement without side effects.

Helicobacter pylori is an aerobic Gram-negative bacilli with a right helix torso and 4 to 8 flagellas (Owen. 1998), which was first identified in the gastric mucosa by Warren and Marshall in Australia in 1983. It has been known to cause digestive diseases such as gastritis, gastric ulcer, duodenal ulcer, gastric lymphoma and gastric cancer. The path of infection of Helicobacter pylori has not yet been determined, but the poorer the poorer the hygiene, the higher the infection rate. Helicobacter pylori infection is one of the most common human infections, with more than 50% of the world's population infected. In particular, more than 20% of 8-year-old children are infected in Korea, and more than 70% after 20s. The infection rate and asymptomatic carriers caused by Helicobacter pylori are much higher than in the country.

As a bacteriological feature, small colonies can be observed by incubating the bacteria in the incubator for 3 to 5 days, and have strong motility and enzyme production ability such as urease, phospholipase A2 and glutamyltranspeptilase. Helicobacter pylori breaks down urea using urease to produce ammonia. Catalase and phospholipase A2 metabolize cell membrane lipids and convert lecithin into lysolecithin to destroy cell membrane function.

Histological studies of Helicobacter pylori have been shown to bind mainly to gastric epithelial cells and mucus layers, and old Helicobacter pylori-related gastritis is known to play a leading role in inducing atrophic changes. have. Atrophic gastritis is known to progress to gastric cancer in some cases by taking multiple pathways, even if Helicobacter pylori is not directly involved. Helicobacter pylori Although the development of related gastritis to gastric cancer is only part of the story, Helicobacter pylori is considered a clear carcinogen or risk factor. As a result of the introduction of antimicrobial treatments for Helicobacter pylori, many drugs have been administered to eradicate Helicobacter pylori so far, either alone or now, and in combination with trichophytaxis. It is widely used as a prescription. Some success has been achieved with the use of antibiotics, but new strains that are resistant to antibiotics have emerged and other approaches have been found due to the side effects of the drugs used.

Vaccine is the fundamental measure for the treatment of future Helicobacter pylori infection, but it is still only a laboratory study and has not been commercialized as an effective vaccine. to be. In recent years, non-antibiotic substances have attracted interest as part of a new treatment concept for Helicobacter pylori that does not use antibiotics.

Therefore, it is necessary to pay attention to the production and utilization of specific antibodies using Helicobacter pylori bacteria as an antigen using an immune system technique or an antigen antibody response. Antibodies as passive immunosuppressive agents have protection against digestive tract infections in those who are unable to produce antibodies due to their poor immune function, such as newborns or children with congenital or acquired immunodeficiency syndrome, and also have reduced immune function due to drugs, malnutrition, and aging. It is also an advantage for adults and children. There have been studies using eg yolk antibodies as a method for inhibiting the attachment of Helicobacter pylori and desorbing already attached bacteria. In particular, the specific antibody IgY to Helicobacter pylori is also involved in inhibiting Helicobacter pylori growth. It is reported. And today, eggs are being released in the form of dietary supplements to eradicate Helicobacter pylori and reduce infection rates, but people tend to avoid eating them because they need to be eaten in the form of high cholesterol.

The present invention has been made to solve the above problems, an object of the present invention, immunization containing a specific antibody by immunizing cows by a method using the characteristics of the antigen-antibody reaction to the helicobacter pylori, the causative agent of gastritis, gastric ulcer Produce milk to be used as a health supplement without side effects.

To this end, the inventors of the present invention examined the ability to produce anti- H. Pylori antibodies in milk produced after immunization with cows using Helicobacter pylori, which is known as a causative agent of digestive diseases such as gastritis, gastric ulcer, gastric ulcer and gastric cancer. The relationship between vaccine dose and antibody production, antigenic specificity, H. pylori bacterial cohesion, acid and heat stability of the antibody, and the effect of vaccination on cow were examined and verified.

The present invention for achieving the above object is characterized in that the immune milk, including the anti-Heliobacter pylori ( Helicobacter pylori ) antibody obtained in a milking cow injected with a vaccine prepared using Helicobacter pylori as an antigen.

The present invention also provides

Passage the Helicobacter pylori strains at 37 ° C. in a 10% CO ₂ incubator at 2-3 days with a sheep blood agar (SBA) plate;

Culturing the above strain in liquid at 37 ° C. for 48 hours in a Mueller Hinton Broth (MHB) medium containing 5% FBS (fetal bovine serum) inactivated at 56 ° C. for 30 minutes;

Sterilization with 0.5% formalin solution after the culture;

Recovering the bacteria by centrifugation at 4,000 rpm for 30 minutes at 4 ° C;

Centrifuging the recovered bacteria with sterile PBS (phosphate buffered saline, pH 7.2) at 4,000 rpm for 30 minutes at 4 ° C., followed by washing and sonicating;

Preparing a vaccine by emulsifying the antigen in 1: 1 ratio with an antigen solution adjusted to O.D. = 1.0 at 667 nm with a spectrophotometer;

Injecting the prepared antigen into the milking cow two or more times at intervals of two weeks;

Milking from the milking cow; characterized in that it comprises a method for producing an immune milk, including an anti- Heliobacter pylori antibody, and the immune milk obtained thereby.

The inventors of the present invention examine the ability of anti- H. Pylori antibodies in milk produced after immunization to cows using Helicobacter pylori as an antigen by the above method, and the relationship between vaccine dose and antibody production, antigen antibody The specificity of, the cohesiveness of H. pylori bacteria, the stability of the acid and heat of the antibody, and the effect of vaccination on dairy cows were examined and verified.

1. Antigen preparations

H. Pylori (KNTC12083) strain was obtained from Korea Gene Bank and subcultured in sheep blood agar (SBA) plate at 37 ° C and 10% CO ₂ incubator every 2 to 3 days. Strains activated by passage culture were incubated for 37 h and 48 h in MHB (Mueller Hinton Broth) medium containing 5% fetal bovine serum (FBS) inactivated at 56 ° C. for 30 minutes. After incubation, sterilization (3hr) with 0.5% formalin solution and centrifuged at 4,000rpm for 30 minutes at 4 ℃ was used to sterilize the recovered bacteria for 30 minutes at 4,000rpm at 4 ℃ with sterile phosphate buffered saline (PBS, pH 7.2). After washing three times by centrifugation, sonicate (pulse 20, duty cycle 50) on ice on for 5 minutes. Sonicate strain was adjusted to OD = 1.0 at a wavelength of 667nm and used as an antigen.

2. Public animals

Twelve cows of Holstein (Holstein), which are being raised as a general specification by the Gyeongsangbuk-do Institute of Animal Science and Technology, were placed into three groups, one each from the beginning, middle, and late stages, as shown in Table 1, and the vaccine doses were 10ml and 20ml, respectively. And divided into 30ml. The feed was fed compound feed produced by Nonghyup Andong Combined Feed Plant Co., Ltd. and hay and water were vegan.

3. Immunity

Vaccines were administered to lactating cows ranging from acetic acid to tertiary tea, and a total of 4 immunizations were performed. Primary immunization was performed by emulsifying H. pylori antigen with Freund's incomplete adjuvant (Sigma F-5881) at a ratio of 1: 1 (5,000 rpm, 5 min) and then 10 ml, 20 ml, on the scapula muscles of the cow. 30 ml were intramuscularly divided into four equal portions. Tertiary immunization was immunized with antigen and Freund's complete adjuvant (Sigma F-5506) in the same manner every two weeks, and the fourth vaccine was immunized in the same manner three weeks after the third vaccine.

4. Serum and Milk Sample Pretreatment

Milk milking was used in tandem semi-automatic milking machine twice a morning and afternoon, and samples for milk analysis were taken 100ml per individual during milking at weekly intervals. The collected milk sample is centrifuged at 14,000 rpm for 30 minutes at 4 ° C to remove solids, and adjusted to pH 4.6 with 1N HCl to remove casein. Casein-depleted whey was adjusted to pH 7.0 with 1N NaOH, stored at -20 ° C, and used for ELISA analysis. The remaining whey was separated and purified for SDS-PAGE and Western blotting experiments. It was.

Blood was collected from 0 to 14 weeks at 2 week intervals, and once collected, about 10 ml was collected from the jugular vein and allowed to stand at room temperature until coagulated. The serum was separated by centrifugation (3,000 rpm, 4 ° C., 15 min). 1 ml each was stored at -20 ℃.

5. Determination of Antibody Titers

Anti-H. In order to determine the relative concentration and titer of milk and serum antibodies against pylori , immunoassay was performed by ELISA (enzyme-linked immunosorbent assay).

Sterilized H. pylori with 0.5% formalin solution (3hrs), washed three times with PBS (phosphate buffer saline, pH 7.2) solution, and then treated with Sonicate (pulse 20, duty cycle 50) at 405 nm in coating buffer (50mM carbonate buffer). , pH 9.6) was adjusted to OD = 0.5 and then coated 100μL each immunoglobulin 96 well plate and overnight (4 ℃). For antigen coating, bovine IgG (Sigma A0706 whole molecule AP conjugate) was coated with a standard. Wash each well three times with washing buffer (10mM PBS, pH 7.4, 0.5% Tween 20), add 150µl of blocking solution (PBS pH 7.4, 5% skim milk), and leave at room temperature for 30 minutes or more. Wash three times with. Casein-depleted whey is diluted in a wash buffer, and then 100 μl of each well is left at room temperature for 2 hours. After washing, dilute anti-bovine IgG (whole molecule) alkaline phosphatase conjugate (Sigma A0705) as a secondary antibody (1: 30,000) in a wash buffer, add 100 µl to the well and leave at room temperature for 1 hour. After washing three times, 100 μl of substrate solution (10% diethanolamine buffe, 1% phosphatase substrate (Sigma 104)) was added thereto, and after 30 minutes, the reaction was stopped with 50 μl of reaction stopper (3N NaOH) and absorbance was measured at 405 nm. .

6. Separation and Purification of Antibodies

1) Isolation of Antibodies from Milk

The milk sample was centrifuged at 14,000 rpm for 30 minutes at 4 ° C to remove fat, dissolved rennin (2 mg / liter) and calcium carbonate (4 g / liter), and incubated at 30 ° C until a curd was formed. To precipitate casein. The precipitated casein was centrifuged at 3,000 rpm for 10 minutes at room temperature, and the precipitate was repeated three times with 35 ml of ammonium sulfate ((NH ₄ ) ₂ SO ₄ ) saturated in 65 ml of the supernatant. The precipitated protein was dialyzed in borated-bufferd saline (BBS, pH 8.4) with a dialysis membrane and lyophilized.

2) Isolation of Antibodies from Serum

10 ml of PBS was added to 10 ml of serum obtained by centrifuging the blood from the cow jugular vein at 3,000 rpm for 15 minutes at 4 ° C, and then 20 ml of saturated milk ((NH ₄ ) ₂ SO ₄ ) was slowly added for 30 to 60 minutes. After stirring, the mixture was centrifuged at 6,000 rpm for 15 minutes. The precipitate was dissolved in PBS and the total amount was 10 ml. Again, the saturated oil was 20%, and the precipitate formed by centrifugation was removed to recover the supernatant. 30% saturated oil was added to the supernatant, and centrifugation was repeated twice. The precipitate was dissolved in a small amount of PBS and desalted by dialysis.

3) Anti- H. pylori Purification and Fractionation of Antibodies

To purify the specific anti- H. Pylori antibody, 79 mg / 2ml of the antibody isolated from milk was dissolved in PBS, and then the anti- H . Pylori antibody was dissolved in 79mg / 2ml PBS, and then 45ml of Bio-Gel A1. Fractions were collected while passing the antibody at a flow rate of 2 ml / min in a column (ψ2.0 × 25㎝) filled with 5m Gel. The absorbance was measured at 280 nm to measure the amount of antibody protein passed through the column, and ELISA was measured at OD = 405 nm to fractionate specific anti- H. Pylori antibodies.

7. Anti- H. pylori  Antibody Antigen Characterization

1) SDS-Polyacrylamide gel electrophoresis

H. pylori molecular weight measured to investigate the properties of the fungus and the anti- H. pylori antibody was carried out by SDS-PAGE (Polyacrylamide gel electrophoresis) . A 4.0% (v / v) stacking gel containing 0.5M Tris-HCl, 10% SDS and 30% acrylamide was used. The separating gel was 1.5M Tris-HCl and 10% SDS, 10% (v / v) gel with 30% acrylamide was used. 30 μg / μl of the purified antibody protein (DW) was diluted 1: 4 with sample buffer and 20 μl were loaded into each well. Sonicated H. pylori bacteria 40㎍ / ㎖ (DW) was diluted with sample buffer 1: 1 and loaded 20 ㎕ each. Marker loaded 5 μl of wide range (Sigma M4038) and low range (Sigma M3913). After electrophoresis, protein bands were stained with Coomassie blue.

2) Confirmation of specificity of antigen by Western blotting

Western blotting was performed to determine whether the anti- H. Pylori antibody produced by the H. pylori antigen responds specifically to the antigen. The whole cell of H. pylori antigen was disrupted by sonicator, and the supernatant obtained by centrifugation (4,000rpm, 5min) was diluted with 80µl / 20µl (sample buffer), 20µl loaded in each well, and NC electrophoresis (Nitrocellulose) was completed after electrophoresis. , Gelman Sciences co.) Transfer the milk antibody and the primary antibody obtained by serum purification. The secondary antibody was reacted by diluting the anti-bovine IgG (whole molecule) alkaline phosphatase conjugate (Sigma A0705) to 1: 30,000 and reacting for 3 minutes with an alkaline phosphatase substrate solution to identify antigenic material on the X-ray film.

8. Anti- H. pylori Physicochemical Properties of Antibodies

1) Anti- H. pylori By antibody H. pylori Fungal aggregation reaction

H. pylori was adjusted to OD 1.0 at 405 nm with PBS and incubated at room temperature for 2 hours after 100 μl coating per well. Whey containing Anti- H. Pylori antibody was diluted 10-fold, and 100 µl was dispensed for each well. After incubation for 2 hours, the degree of aggregation was observed under a × 500-fold optical microscope.

2) Anti- H. pylori Stability test for heat and acid of antibody

After the antibody was produced, the milk sample was collected and centrifuged (14,000 rpm, 4 ° C., 30 min) to remove solids, adjusted to pH 4.6 with 1N HCl to remove casein, and adjusted to pH 7.0 with 1N NaOH. whey) was used.

To find out the stability of heat with the addition of additives, add 150 ml of 0.5 ml centrifuge tube to whey, 20% maltose, 20% glycerol and 0.2% glutamic acid, and add a heating block (model: HF-21, YAMATO SCIENTIFIC CO. LTD) was treated at 60 ° C, 70 ° C, 80 ° C, 100 ° C and 135 ° C by hour and then anti-H. Pylori antibody activity was measured.

The stability test for acid was adjusted to pH = 7.0 ~ 1.0 with 1N HCl, and alkaline state was adjusted to pH = 8 ~ 12 with 1N NaOH to measure anti- H. Pylori antibody activity by ELISA.

9. Rectal Temperatures

After the vaccination, the rectal temperature was measured at 0h, 3h, 6h, 12h, 24h and 48h intervals after the vaccination using a thermometer to examine the changes in the cows due to vaccination.

10. Statistical Analysis

Statistical analysis of the experimental results was carried out by covariance analysis using SPSS (10.0 for windows), and the significance of each treatment group was recognized when p <0.05.

11. Experimental results

end. H. Pylori culture

When H. pylori was subcultured to SBA (sheep blood agar plate) medium As shown in Fig. 1, a characteristic colorless and transparent colony was formed, and the results of urease test were shown. As positive as 2, it was determined as H. Pylori and used as an antigen.

Fig. One. H. pylori culture

Fig. 2. Urea test

I. Anti-according to vaccine dose H. pylori Antibody content

1) Anti-blood H. pylori Antibody Content Change

Fig. Figure 3 shows the change in the anti- H. Pylori antibody content in the blood according to the vaccine dose. Overall, the anti- H. Pylori antibody content in the blood was increased from 2 weeks after vaccination. Significant differences were found with the control. In particular, the maximum production was seen at 6 weeks, and lasted up to 14 weeks (p <0.05). This is similar to the results of the study that the antibody production in the serum following vaccination sharply increases after 2 weeks of vaccination. Six weeks after vaccination, 0.78 mg / ml for the 10 ml group, 1.05 mg / ml for the 20 ml group and 0.73 mg / ml for the 30 ml group were 2.1 times, compared to the control group with an average antibody content of 0.38 mg / ml. It increased by 2.8 times and 1.9 times. Unlike the study results in which the vaccine dose did not show a significant difference in the amount of antibody production, the 20 ml vaccination produced the most antibody in the order of 20 ml> 10 ml> 30 ml vaccination (p <0.05).

2) Anti- in milk H. pylori Antibody Content Change

Fig. Figure 4 shows the change of anti- H. Pylori antibody content in whey according to vaccine dose. Most of the immunogenic proteins in whey are known to be transported from serum or synthesized in mammary glands. Thus, in this experiment, the production of antibody in whey was found to increase in a similar trend to serum. Whey antibody content also started to increase from 2 weeks and showed maximum production at 6 weeks. After 6 weeks, the average antibody production was 4.62 ㎍ / ml for the control, 13.8 ㎍ / ml for the 10 ml vaccine, and 15.1 for the 20 ml. The administration of ㎍ / ㎖, 30 ㎖ was 14.6 ㎍ / ㎖ showed a significant difference from the control (p <0.05). However, there was no significant difference in the amount of antibody produced between treatment groups according to vaccine dose (P> 0.05). Therefore, considering the amount of antibody production and the stress of cows, 10 ml of vaccine seems to be the most appropriate.

All. Anti- H. pylori Antibody Characteristics

1) Anti- H. pylori Specificity of Antibody

SDA-PAGE was performed to analyze the isolated anti- H. Pylori antibody and antigen H. pylori (Fig. 5). There are many kinds of antibody proteins that can cause an immune response depending on the type of antigen, but antibodies in bovine serum and whey are classified into four types, IgG1, IgG2, IgM, and IgA, depending on the characteristics of the antigen response. . IgG antibodies are large molecules of about 150 kDa consisting of two polypeptides. The heavy chain of about 50 kDa and the light chain of 25 kDa are composed of light chains, and Ig (immunoglobulin) is composed of two identical heavy and light chains.

In this experiment, anti- H. Pylori antibody showed the same pattern as that reported by Kim and Shin et al., When one band was formed, which is shown as a light chain at a heavy chain of 50kDa and a molecular weight of 24kDa.

Fig. 5.SDS-PAGE patterns of antibody from diary cows immunized with H. pylori .

      M: Marker (20kDa: trypsin inhibitor, 29kDa: carbonic anhydrase, 36kDa: glyceraldehyde- 3-phosphate dehydrogenase, 45kDa: ovalbumin, 66kDa: albumin, bovine serum, 116kDa: β-galactosidase, 205kDa: Myosin)

        A: Purification IgG from milk of hyper-immunized diary cows.

2) specificity of the antigen

Fig. Figure 6 shows the results of Western blotting to determine the specificity of the antigen caused by anti- H. Pylori antibody. As a result of molecular weight measurement by SDS-PAGE of the antigen H. pylori , the antigenic protein formed 12 bands. Western blotting using serum, serum tablets, whey, and whey tablets with primary antibodies among 12 proteins revealed seven antigenic compounds, of which the primary antigenic compounds had molecular weights of 97, 66, and 34 kDa. Three bands were identified.

Fig. 6.Western blotting of purified antibody from dairy cows immunized with H. pylori.

    Primary antibody was serum (line A), purified serum (lineB), whey (lineC) and purified whey (lineD).

E: Sonicated H. pylori whole cell antigen

     M: Marker (20kDa: trypsin inhibitor, 29kDa: carbonic anhydrase, 36kDa: glyceraldehyde- 3-phosphate dehydrogenase, 45kDa: ovalbumin, 66kDa: albumin, bovine serum, 116kDa: β-galactosidase, 205kDa: Myosin.

3) Anti -H. pylori Purification and Fractionation of Antibodies

Bio-Gel A-1.5m Gel was passed to purify and fractionate specific anti- H. Pylori antibodies from antibodies isolated from milk. The absorbance was measured at 280 nm to measure the protein amount of the antibody protein fraction that was passed, and ELISA was measured at 405 nm to determine the antibody content (Fig. 7).

Passing through the column revealed that most of the antibody proteins were contained between fractions 9-30 and specific anti- H. Pylori antibodies were located between fractions 17-25. Therefore, specific anti- H. Pylori antibodies could be obtained by separating proteins between fractions 17-25.

la. Anti- H. pylori Physicochemical Characteristics of Antibodies

1) flocculation reaction

Aggregation reaction refers to a phenomenon in which antigen particles having a certain size, such as bacteria and red blood cells, react with the antibody to form aggregates.

The degree of aggregation of the antibody against the antigen is shown in Fig. In the control non-immune, such as 8, while not causing agglutination anti- H. pylori antibody immunized with H. pylori bacteria has caused a strong aggregation response against H. pylori bacteria. From what already reported IgY specific antibodies were found to inhibit H. pylori growth shows a high disinfecting power in whey antibody obtained by immunization with H. pylori bacteria has four types of reports. In this experiment, anti- H. Pylori antibodies in whey showed a high aggregation rate of 1/10 against H. pylori (Table 2). As a result of this flocculation reaction, ingestion of cows may be effective in treating and preventing H. pylori infection.

      <Control> <Treatment>

Fig. 8.Agglutination of antigen by anti- H. pylori  antibody

2) Stability of Acid Treatment of Antibodies

Whey, from which casein was removed from milk, was measured for anti- H. Pylori antibody content at pH 1-12 (Fig. 9). The anti- H. pylori antibody activity showed 100% activity in the pH range of 5-10, but decreased to 70% at pH 4 and pH 11. Almost no activity was observed at pH 1-2 and pH 12. These results showed the same trend as the conventional research report on the stability of IgY in egg yolk, and the antibodies in whey also remained relatively stable against acids and alkalis.

3) Stability against Heat Treatment of Antibodies

Milk heat treatment changes the functional properties of milk and is widely used in the production of various dairy products. This heat treatment affects the milk composition and thus affects the sensory and nutritional quality of the milk, which is important for the final product properties. Changes in ingredients due to heat treatment in milk are known to affect heat-induced interactions and stability of whey proteins.

According to the previous research reports that sugar solution was added to IgG and IgY, it was tested for heat stability by adding 20% maltose, 20% glycerol, and 0.2% glutamic acid. Fig. Same as 10 At 60 ° C., all of the various additives were stable for 60 minutes, and at 70 ° C., activity began to drop over time, and after 60 minutes, the activity decreased by 40%. The activity was 77% at 80 ° C. for 4 minutes, and the activity dropped over time, and after 8 minutes, no activity was observed. It was relatively stable at 100 ° C. for 1 minute, but the activity dropped sharply after 1 minute and was almost inactive after 3 minutes. Even at 135 ° C., the activity dropped more than 50% within 1 minute, and almost no activity appeared after 2 minutes.

Heat denaturation of whey protein by electrophoresis was found to be heat-safe in order of immunoglobulin, serum albumin, β-lactoglobulin, and α-lactalbumin, and 11%, 48%, 68%, As in the previous study, which decreased by 95%, it was relatively stable in this experiment.

When IgY was heated for 1 minute, the relative antibody value was 87% at 70 ° C and 70% at 75 ° C, but little activity was observed at 90 ° C. Compared to IgY, the milk is more stable than IgY when the results show that the stability for 1 minute even at 100 ℃. In addition, it is reported that depending on the additive, the antibody affects the stability to heat. However, the addition of additives (20% maltose, 20% glycerol, 0.2% glutamic acid) to whey did not significantly affect the thermal stability. Unlike other reports, this is considered to be different from this experiment due to the difference in heat treatment time and temperature.

In view of the heat stability results, anti- H. Pylori antibody activity can be significantly maintained by pasteurization (62 ℃, 30 minutes) during milk sterilization by heat treatment.

hemp. Effect of Vaccine on Cows

1) Changes in oil production following vaccination

In order to determine the changes in the milk flow after the vaccine administration, the average daily oil flow was examined from the first 3 days before the vaccine to the 6 days after the vaccine. Overall, the amount of oil started to decrease from the day of vaccine administration, and the average decrease was 12% on the first day after vaccination, and the recovery started on the fourth day and recovered more than 95% on the sixth day.

Vaccine administration had an effect on the change of acid flow rate. After 1 day of vaccination, the change of acid flow rate decreased by 3% for 10ml, 17% for 20ml, and 6% for 30ml. There was a slight difference in the recovery of oil production according to the time of lactation or individual characteristics, but it took up to one week.

The main cause of the change in the flow rate according to the vaccine dose was considered to be due to the stress caused by the vaccine administration, and 10ml vaccine administration had the least effect on the milk production. In addition, the control group also showed a decrease in the milk production, which is thought to have a greater effect due to the stress of vaccine administration and blood collection.

2) Changes in body temperature of cows after vaccination

Fig. Figure 11 shows the change in the rectal temperature of the cow after vaccination, compared with the control group, which showed a rise in body temperature within the normal range. Contrary to previous reports that no rectal temperature change occurred after vaccination, control body temperature was 39.3 ℃ and vaccine dose was 39.1 ℃ after vaccination. Normal temperature was maintained. And 6 hours after vaccination, both the control and the treatments showed an increase in body temperature, which is judged to be due to the change in the biorhythm of the cow by one crossing.

12. Conclusion

This study examined the ability of anti- H. Pylori antibodies in milk produced after immunization to cows with Helicobacter pylori , a known cause of digestive diseases such as gastritis, gastric ulcer, gastric ulcer and gastric cancer. And the relationship between antibody production, antigenic specificity, H. pylori bacterial cohesion, acid and heat stability, and the effect of vaccination on cows.

(1) The anti- H. Pylori antibody content in serum and whey according to the vaccine dose was higher than that of the control in all 10ml, 20ml and 30ml vaccines. There was no significant difference, but 20 ml administration showed the highest antibody production. Anti- H. Pylori antibody production in whey according to vaccine dose was similar to that in serum.

(2) As a result of SDS-PAGE of anti- H. Pylori antibody, heavy chain was about 50kDa and light chain was about 24kDa.

(3) As a result of molecular weight measurement of H. pylori antigen protein, 12 bands were formed. Western blotting was performed to determine the antigenic specificity of Anti- H . Pylori. Seven antigenic substances were identified in serum, serum, whey and whey. The main antigenic substances were 97, 66 and 34kDa. .

(4) As a result of the coagulation reaction, anti- H . Pylori antibody in whey showed 1/10 coagulation value against H. pylori .

(5) Anti- H. Pylori antibody showed stable activity against acid and alkali in the range of pH 5 ~ 10 and showed 100% activity.

(6) The anti- H. Pylori antibody was stable for 60 minutes at 60 ° C and relatively stable at 70 ° C, but activity decreased by 40% after 60 minutes. 77% activity was maintained at 80 ° C. for 4 minutes, and relatively stable at 100 ° C. for 1 minute.

(7) The flow rate decreased by 12% due to vaccination and recovered for up to 1 week.

(8) As a result of measuring the body temperature of the cow after vaccination, the body temperature increased within the normal range compared to the control.

According to the present invention of the above configuration, by the method using the characteristics of the antigen-antibody reaction H. pylori , the causative agent of gastritis, gastric ulcers to the cow to produce immune milk containing specific antibodies as a dietary supplement without side effects It becomes available.

In addition, we demonstrated the possibility of the high production of anti- H. Pylori antibodies in milk produced after immunization with cows using Helicobacter pylori as an antigen, and additionally, the relationship between vaccine dose and antibody production , The specificity of the antigenic antibody, the cohesiveness of H. pylori bacteria, the stability of the antibody against acid and heat, and the effect of vaccination on cows were verified.

Claims (3)

delete Passage the Helicobacter pylori strains at 37 ° C. in a 10% CO ₂ incubator at 2-3 days with a sheep blood agar (SBA) plate; Culturing the above strain in liquid at 37 ° C. for 48 hours in a Mueller Hinton Broth (MHB) medium containing 5% FBS (fetal bovine serum) inactivated at 56 ° C. for 30 minutes; Sterilization with 0.5% formalin solution after the culture; Recovering the bacteria by centrifugation at 4,000 rpm for 30 minutes at 4 ° C; Centrifuging the recovered bacteria with sterile PBS (phosphate buffered saline, pH 7.2) at 4,000 rpm for 30 minutes at 4 ° C., followed by washing and sonicating; Preparing a vaccine by emulsifying the antigen in 1: 1 ratio with an antigen solution adjusted to O.D. = 1.0 at 667 nm with a spectrophotometer; Intramuscularly injecting the prepared antigens two or more times up to eight times at two week intervals in the milking cow; Milking from the milking cow; characterized in that it comprises a Method for producing immune milk containing anti- Helicobacter pylori antibody. Produced by the method of claim 2 Immune milk, including anti- Heliobacter pylori antibodies.